A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort.

Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

A ligand-independent origin of abscisic acid perception.

Land plants are considered monophyletic, descending from a single successful colonization of land by an aquatic algal ancestor. The ability to survive dehydration to the point of desiccation is a key adaptive trait enabling terrestrialization. In extant land plants, desiccation tolerance depends on the action of the hormone abscisic acid (ABA) that acts through a receptor-signal transduction pathway comprising a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module. Early-diverging aeroterrestrial algae mount a dehydration response that is similar to that of land plants, but that does not depend on ABA: Although ABA synthesis is widespread among algal species, ABA-dependent responses are not detected, and algae lack an ABA-binding PYL homolog. This raises the key question of how ABA signaling arose in the earliest land plants. Here, we systematically characterized ABA receptor-like proteins from major land plant lineages, including a protein found in the algal sister lineage of land plants. We found that the algal PYL-homolog encoded by Zygnema circumcarinatum has basal, ligand-independent activity of PP2C repression, suggesting this to be an ancestral function. Similarly, a liverwort receptor possesses basal activity, but it is further activated by ABA. We propose that co-option of ABA to control a preexisting PP2C-SnRK2-dependent desiccation-tolerance pathway enabled transition from an all-or-nothing survival strategy to a hormone-modulated, competitive strategy by enabling continued growth of anatomically diversifying vascular plants in dehydrative conditions, enabling them to exploit their new environment more efficiently.

Protamines from liverwort are produced by post-translational cleavage and C-terminal di-aminopropanelation of several male germ-specific H1 histones.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.

Triazine Probes Target Ascorbate Peroxidases in Plants.

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), liverwort (Marchantia polymorpha), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in apx1 mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.

Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts.

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

Phototropin perceives temperature based on the lifetime of its photoactivated state.

Living organisms detect changes in temperature using thermosensory molecules. However, these molecules and/or their mechanisms for sensing temperature differ among organisms. To identify thermosensory molecules in plants, we investigated chloroplast positioning in response to temperature changes and identified a blue-light photoreceptor, phototropin, that is an essential regulator of chloroplast positioning. Based on the biochemical properties of phototropin during the cellular response to light and temperature changes, we found that phototropin perceives temperature based on the temperature-dependent lifetime of the photoactivated chromophore. Our findings indicate that phototropin perceives both blue light and temperature and uses this information to arrange the chloroplasts for optimal photosynthesis. Because the photoactivated chromophore of many photoreceptors has a temperature-dependent lifetime, a similar temperature-sensing mechanism likely exists in other organisms. Thus, photoreceptors may have the potential to function as thermoreceptors.

Phytofiltration of chosen metals by aquarium liverwort (Monosoleum tenerum).

Aquatic bryophytes are widely used as indicators of water pollution with various substances, including metals. We present a first study concerning the feasibility of Monosoleum tenerum to remove potentially toxic metals Zn, Cu, Ni, Mn and Fe from water. The novel approach to study the bioaccumulation under controlled conditions and in vitro grown aquarium liverworts was applied. The plants were exposed to 1, 10 and 100 ppm multi-metal solution for 7 days and subsequently the metal content was determined in plant material and media to calculate the bioaccumulation factor (BAF). The study revealed various accumulation behavior (metal and dose dependent), which followed the order of Cu > Zn > Mn > Ni > Fe for 1 ppm; Zn > Cu > Mn > Fe > Ni for 10 ppm and Cu > Fe > Zn > Ni > Mn for 100 ppm solution. However, with increasing Cu, Mn and Ni concentration in the solution, BAF decreased. For Zn the highest BAF value (136) was obtained for the 10 ppm solution. Fe bioaccumulation increased with the increasing solution concentration. After 7 week exposure, the results indicated highest accumulation of Cu (3,25 mg) followed by Fe (1,8 mg) in the plant tissue for the 100 ppm solution. Overall, the bryophyte M. tenerum demonstrate high potential for the removal of toxic metals from the multi-metal solution via accumulation in plant tissue. The BAF values were proper to indicate the use of M. tenerum for phytofiltration of waste waters affected by Zn, Cu, Ni, Mn and Fe.

Biological Responses to Cadmium Stress in Liverwort Conocephalum conicum (Marchantiales).

Oxidative damage (production and localization of reactive oxygen species) and related response mechanisms (activity of antioxidant enzymes), and induction of Heat Shock Protein 70 expression, have been studied in the toxi-tolerant liverwort Conocephalum conicum (Marchantiales) in response to cadmium stress using two concentrations (36 and 360 µM CdCl2). Cadmium dose-dependent production of reactive oxygen species (ROS) and related activity of antioxidant enzymes was observed. The expression level of heat shock protein (Hsp)70, instead, was higher at 36 µM CdCl2 in comparison with the value obtained after exposure to 360 µM CdCl2, suggesting a possible inhibition of the expression of this stress gene at higher cadmium exposure doses. Biological responses were related to cadmium bioaccumulation. Since C. conicum was able to respond to cadmium stress by modifying biological parameters, we discuss the data considering the possibility of using these biological changes as biomarkers of cadmium pollution.

Transcriptional and Morpho-Physiological Responses of Marchantia polymorpha upon Phosphate Starvation.

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.

Transformation of Riccia fluitans, an Amphibious Liverwort Dynamically Responding to Environmental Changes.

The colonization of land by streptophyte algae, ancestors of embryophyte plants, was a fundamental event in the history of life on earth. Bryophytes are early diversifying land plants that mark the transition from freshwater to terrestrial ecosystems. The amphibious liverwort Riccia fluitans can thrive in aquatic and terrestrial environments and thus represents an ideal organism to investigate this major transition. Therefore, we aimed to establish a transformation protocol for R. fluitans to make it amenable for genetic analyses. An Agrobacterium transformation procedure using R. fluitans callus tissue allows to generate stably transformed plants within 10 weeks. Furthermore, for comprehensive studies spanning all life stages, we demonstrate that the switch from vegetative to reproductive development can be induced by both flooding and poor nutrient availability. Interestingly, a single R. fluitans plant can consecutively adapt to different growth environments and forms distinctive and reversible features of the thallus, photosynthetically active tissue that is thus functionally similar to leaves of vascular plants. The morphological plasticity affecting vegetative growth, air pore formation, and rhizoid development realized by one genotype in response to two different environments makes R. fluitans ideal to study the adaptive molecular mechanisms enabling the colonialization of land by aquatic plants.

Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes. RESULTS: In this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid. CONCLUSION: PabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.

A bHLH Transcription Factor Regulates Bisbibenzyl Biosynthesis in the Liverwort Plagiochasma appendiculatum.

Liverworts, a section of the bryophyte plants which pioneered the colonization of terrestrial habitats, produce cyclic bisbibenzyls as secondary metabolites. These compounds are generated via the phenylpropanoid pathway, similar to flavonoid biosynthesis, for which basic helix-loop-helix (bHLH) transcription factors have been identified as one of the important regulators in higher plants. Here, a bHLH gene homolog (PabHLH) was isolated from the liverwort species Plagiochasma appendiculatum and its contribution to bisbibenzyl biosynthesis was explored. Variation in the abundance of PabHLH transcript mirrored that of tissue bisbibenzyl content in three different liverwort tissues. A phylogenetic analysis based on the bHLH domain sequence suggested that the gene encodes a member of bHLH subgroup IIIf, which clusters proteins involved in flavonoid synthesis. The gene's transient expression in onion epidermal cells implied that its product localized to the nucleus, and a transactivation assays in yeast showed that it was able to activate transcription. In both callus and thallus, the overexpression of PabHLH boosted bisbibenzyl accumulation, while also up-regulating PaPAL, Pa4CL1, PaSTCS1 and two genes encoding P450 cytochromes, and its RNA interference (RNAi)-induced suppression down-regulated the same set of genes and reduced the accumulation of bisbibenzyls. The abundance of PaCHS and PaFNSI transcript was related to flavonoid accumulation in transgenic thallus. PabHLH represents a candidate for the metabolic engineering of bisbibenzyl content.

Diversity of Secondary Metabolites in the Liverwort Syzygiella rubricaulis (Nees) Stephani (Jamesoniellaceae, Marchantiophyta) from Neotropical High Mountains.

In order to evaluate the chemical diversity of Syzygiella rubricaulis (Nees) Stephani, a species with a disjunct distribution in the neotropical high mountains, a phytochemical study was carried out with samples from 12 different populations of different altitudes from four South American countries. The chemical profiles of lipophilic extracts were analyzed by GC/MS for each population and 50 different compounds were found with the predominance and richness of sesquiterpenes. The majority of the compounds were found only in one population and the total number of substances ranged from 1 to 15 among the populations, but these numbers were not correlated with altitude, and characterize each population as distinct, based on similarity analysis. The qualitative and quantitative variations of metabolites found are a response to different conditions, under which they live, mostly likely altitudinal conditions. Further studies on the quantification of these chemicals may provide information on their ecological roles and importance for the distribution of S. rubricaulis at different altitudes. Despite the known richness of secondary metabolites produced by bryophytes, they are still poorly explored in the context of the ecological expressions.

Land plants drive photorespiration as higher electron-sink: comparative study of post-illumination transient O2 -uptake rates from liverworts to angiosperms through ferns and gymnosperms.

In higher plants, the electron-sink capacity of photorespiration contributes to alleviation of photoinhibition by dissipating excess energy under conditions when photosynthesis is limited. We addressed the question at which point in the evolution of photosynthetic organisms photorespiration began to function as electron sink and replaced the flavodiiron proteins which catalyze the reduction of O2 at photosystem I in cyanobacteria. Algae do not have a higher activity of photorespiration when CO2 assimilation is limited, and it can therefore not act as an electron sink. Using land plants (liverworts, ferns, gymnosperms, and angiosperms) we compared photorespiration activity and estimated the electron flux driven by photorespiration to evaluate its electron-sink capacity at CO2 -compensation point. In vivo photorespiration activity was estimated by the simultaneous measurement of O2 -exchange rate and chlorophyll fluorescence yield. All C3-plants leaves showed transient O2 -uptake after actinic light illumination (post-illumination transient O2 -uptake), which reflects photorespiration activity. Post-illumination transient O2 -uptake rates increased in the order from liverworts to angiosperms through ferns and gymnosperms. Furthermore, photorespiration-dependent electron flux in photosynthetic linear electron flow was estimated from post-illumination transient O2 -uptake rate and compared with the electron flux in photosynthetic linear electron flow in order to evaluate the electron-sink capacity of photorespiration. The electron-sink capacity at the CO2 -compensation point also increased in the above order. In gymnosperms photorespiration was determined to be the main electron-sink. C3-C4 intermediate species of Flaveria plants showed photorespiration activity, which intermediate between that of C3- and C4-flaveria species. These results indicate that in the first land plants, liverworts, photorespiration started to function as electron sink. According to our hypothesis, the dramatic increase in partial pressure of O2 in the atmosphere about 0.4 billion years ago made it possible to drive photorespiration with higher activity in liverworts.

Constituents of the Argentine Liverwort Plagiochila diversifolia and Their Insecticidal Activities.

Three new compounds, a bicyclogermacrene (1) and two 2,3-secoaromadendrane esters (2 and 3), together with (13S)-13-hydroxylabda-8,14-diene (4), fusicogigantone B (5), 3α,14-diacetoxy-2-hydoxybicyclogermacrene (6), fusicogigantone A (7), neofuranoplagiochilal (8), plagiochiline B (9), furanoplagiochilal (10), trans-nerolidol, spathulenol, α-tocopherol, and (+)-globulol were isolated from an Argentine collection of Plagiochila diversifolia. Their structures were elucidated by extensive mono and bidimensional NMR studies. Compounds 4, 5, and 6, incorporated to the larval diet at 100 µg per g of diet, reduced the larval growth of Spodoptera frugiperda (Lepidoptera: Noctuidae) by 70 ± 25, 57 ± 23, and 33 ± 16%, respectively. Compounds 4 and 5 produced 70% and 60% larval mortality at early instars. The latter also showed antifeedant properties in the Choice Test, with a feeding ratio of 0.54 ± 0.16.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

MpMLO1 controls sperm discharge in liverwort.

In bryophytes, sexual reproduction necessitates the release of motile sperm cells from a gametophyte into the environment. Since 1856, this process, particularly in liverworts, has been known to depend on water. However, the molecular mechanism underlying this phenomenon has remained elusive. Here we identify the plasma membrane protein MpMLO1 in Marchantia polymorpha, a model liverwort, as critical for sperm discharge from antheridia. The MpMLO1-expressing tip cells among the sperm-wrapping jacket cells undergo programmed cell death upon antheridium maturation to facilitate sperm discharge after the application of water and even hypertonic solutions. The absence of MpMLO1 leads to reduced cytoplasmic Ca2+ levels in tip cells, preventing cell death and consequently sperm discharge. Our findings reveal that MpMLO1-mediated programmed cell death in antheridial tip cells, regulated by cytosolic Ca2+ dynamics, is essential for sperm release, elucidating a key mechanism in bryophyte sexual reproduction and providing insights into terrestrial plant evolution.